EP1106690B1 - Endometriose-assoziierte-Autoantigene kodierendes Polynukleotid - Google Patents

Endometriose-assoziierte-Autoantigene kodierendes Polynukleotid Download PDF

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EP1106690B1
EP1106690B1 EP00310408A EP00310408A EP1106690B1 EP 1106690 B1 EP1106690 B1 EP 1106690B1 EP 00310408 A EP00310408 A EP 00310408A EP 00310408 A EP00310408 A EP 00310408A EP 1106690 B1 EP1106690 B1 EP 1106690B1
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Prior art keywords
repro
seq
polypeptide
sequence
antibody
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EP1106690A2 (de
EP1106690A3 (de
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A. Said El Shami
Surendra Nath Menon
Cynthia K. French
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Siemens Medical Solutions Diagnostics Inc Canada
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Diagnostic Products Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity

Definitions

  • This invention is directed to the field of molecular biology in general, and, more specifically, to a polypeptide associated with endometriosis, an isolated polynucleotide encoding the polypeptide, and methods of using these molecules.
  • Endometriosis is a painful disorder that is characterized by the ectopic implantation of functioning endometrial tissue into the abdominal wall and the outer surface of various organs including, most commonly, the lower bowel, ovaries and fallopian tubes.
  • endometriosis-specific genes have not been identified and the events relating to the development of endometriosis are poorly understood.
  • several reports suggest that retrograde menstruation linked with abnormal immune function may play a role in establishing ectopic endometrium lesions.
  • Many attempts to isolate antigens from ectopic endometrium lesions have failed, due to the necrotic nature of the lesions.
  • WO 94/28021 describes isolation of an endometrial antigen from female patients with endometri
  • Endometriosis also is recognized as having an autoimmune component.
  • IgG and IgA auto-antibodies that react with multiple endometrial antigens have been documented in patients with endometriosis.
  • attempts to develop IgG or IgA-based assays for the diagnosis of endometriosis has fallen short of fruition.
  • Studies have shown that circulating IgG antibodies that bind multiple endometrial proteins can be detected in women with endometriosis to varying degrees.
  • This invention provides an isolated cDNA molecule and an alternately spliced variant encoding autoantigens associated with endometriosis.
  • the autoantigen is called Repro-EN-1.0.
  • the alternately spliced variant is called IB1.
  • Subjects diagnosed with endometriosis have been found to have antibodies that specifically bind to Repro-EN-1.0 polypeptide and/or a IB1 polypeptide. These antibodies represent a highly sensitive and specific diagnostic marker for endometriosis.
  • Recombinant Repro-EN-1.0 protein and recombinant IB1 protein are useful to detect such antibodies in immunoassays.
  • this invention provides a recombinant polynucleotide comprising a nucleotide sequence encoding polypeptide Repro-EN-1.0 of SEQ ID NO:2. or IB1 of SEQ ID NO:4 wherein the polypeptide specifically binds to antibodies from subjects diagnosed with endometriosis.
  • the nucleotide sequence is selected from the Repro-EN-1.0 sequence of SEQ ID NO:1 or IB1-sequence of SEQ ID NO: 3.
  • the nucleotide sequence is identical to nucleotides 176 to 2755 of SEQ ID NO:1 or nucleotides 176 to 2986 of SEQ ID NO:3.
  • the polynucleotide further comprises an expression control sequence operatively linked to the nucleofide sequence.
  • this invention provides a polynucleotide primer pair which amplifies a nucleotide sequence encoding polypeptide Repro-EN-1.0 or IB1 wherein the polypeptide specifically binds to antibodies from subjects diagnosed with endometriosis.
  • the pair comprises: 1) a 3' primer of at least 7 nucleotides that specifically hybridizes to a 3' end of the nucleotide sequence or downstream from the sequence, and 2) a 5' primer of at least 7 nucleotides that specifically hybridizes to the 3' end of the complement of the nucleotide sequence or downstream from the complement of the sequence.
  • this invention provides a recombinant cell comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence encoding polypeptide Repro-EN-1.0 of SEQ ID NO:2 or IB1 of SEQ ID NO:4, wherein the polypeptide specifically binds to antibodies from subjects diagnosed with endometriosis.
  • this invention provides a purified, recombinant Repro-EN-1.0 polypeptide whose amino acid sequence is identical to that of SEQ ID NO:2, or an allelic variant of SEQ ID NO:2, or an IB1 polypeptide whose amino acid sequence is identical to that of SEQ ID NO: 4, or an allelic variant of SEQ ID No: 4.
  • this invention provides a purified polypeptide comprising Repro-EN-1.0 of SEQ ID NO: 2 or IB1 of SEQ ID NO: 4, wherein the polypeptide specifically binds to antibodies from subjects diagnosed with endometriosis.
  • this invention provides a composition consisting essentially of an antibody that specifically binds to Repro-EN-1.0 polypeptide of SEQ ID NO: 2, or IB1 polypeptide of SEQ ID NO: 4.
  • this invention provides a method of diagnosing endometriosis in a subject.
  • the method comprises the steps of: (a) detecting a test amount of an antibody that specifically binds to Repro-EN-1.0 polypeptidewhose amino acid sequence is identical to SEQ ID NO: 2 or an allelic variant of SEQ ID NO: 2 or IB1 polypeptide whose amino acid sequence is identical to SEQ ID NO: 4 or an allelic variant of SEQ ID NO: 4 in a sample from the subject; and (b) comparing the test amount with a normal range of the antibody in a control sample from a subject who does not suffer from endometriosis.
  • a test amount above the normal range provides a positive indication in the diagnosis of endometriosis.
  • the sample can be a blood product, e.g., serum, peritoneal fluid, menstrual fluid, vaginal secretion or urine.
  • the antibody is an IgE, IgG or IG 4 immunoglobulin.
  • the step of detecting comprises capturing the antibody from the sample with immobilized Repro-EN-1.0 or IB1, and detecting captured antibody.
  • the step of detecting captured antibody can comprise contacting the captured antibody with a detectable antibody that specifically binds immunoglobulins and detecting binding between the captured antibody and the detectable antibody.
  • the step of detecting can comprise capturing the antibody from the sample with an immobilized anti-immunoglobulin antibody and detecting captured antibody.
  • the step of detecting captured antibody can comprise contacting the captured antibody with Repro-EN-1.0 or IB1, and detecting binding between the captured antibody and the Repro-EN-1.0 or IB1.
  • this invention provides a method for use in following the progress of endometriosis in a subject.
  • the method comprises the steps of: (a) detecting first and second amounts of an antibody that specifically bind Repro-EN-1.0 polypeptides whose amino acid sequence is identical to that of SEQ ID NO: 2 or an allelic variant of SEQ ID NO: 2 or IB1 polypeptide whose amino acid sequence is identical to that of SEQ ID NO: 4 or an allelic variant of SEQ ID NO: 4 in samples from the subject at a first and a second time, respectively; and (b) comparing the first and second amounts.
  • An increase between the first and second amounts indicates progression of the endometriosis and a decrease between the first an second amounts indicates remission of the endometriosis.
  • this invention provides a screening method for determining whether a compound increases or decreases the expression of Repro-EN-1 of SEQ ID NO: 2 or IB1 of SEQ ID NO: 4 in a cell, comprising contacting the cell with the compound and determining whether the-production of Repro-EN1.0 mRNA or polypeptide, or IB1 mRNA or polypeptide, is increased or decreased.
  • this invention provides a method of detecting polymorphic forms of Repro-EN-1.0 of SEQ ID NO: 1 or 2 or IB1 of SEQ ID NO: 3 or 4 comprising the steps of: a) determining the identity of a nucleotide or amino acid at a selected position within the sequence of a test Repro-EN-1.0 gene orpolypeptide, or IB1 gene or polypeptide; b) determining the identity of the nucleotide or amino acid at the corresponding position of native Repro-EN-1.0 (gene or polypeptide of SEQ ID NO: 1 or 2 or IB1 gene or polypeptide of SEQ ID NO: 3 or 4) ; and c) comparing the identity from the test gene or polypeptide with the identity of the native gene or polypeptide, whereby a difference in identity indicates that the test gene or polypeptide a polymorphic form of Repro-EN-1.0 or IB1.
  • Polynucleotide refers to a polymer composed of nucleotide units.
  • Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as nucleic acid analogs.
  • Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds.
  • nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.
  • PNAs peptide-nucleic acids
  • Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer.
  • the term “nucleic acid” typically refers to large polynucleotides.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides.
  • nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C)
  • this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • cDNA refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
  • the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences.”
  • “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides.
  • the two molecules can be described as complementary, and furthermore, the con-tact surface characteristics are complementary to each other.
  • a first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide.
  • the polynucleotide whose sequence 5'-TATAC-3' is complementary to a polynucleotide whose sequence is 5'-GTATA-3'.
  • a nucleotide sequence is "substantially complementary" to a reference nucleotide sequence if the sequence complementary to the subject nucleotide sequence is substantially identical to the reference nucleotide sequence.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system.
  • coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings
  • non-coding strand used as the template for transcription
  • a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns
  • Recombinant polynucleotide refers to a polynucleotide having sequences that are not naturally joined together.
  • An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.
  • a host cell that comprises the recombinant polynucleotide is referred to as a "recombinant host cell.”
  • the gene is then expressed in the recombinant host cell to produce, e.g., a "recombinant polypeptide.”
  • a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • “Expression control sequence” refers to a nucleotide sequence in a polynucleotide that regulates the expression (transcription and/or translation) of a nucleotide sequence operatively linked thereto. "Operatively linked” refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part (e.g., transcription of the sequence).
  • Expression control sequences can include, for example and without limitation, sequences of promoters (e.g., inducible or constitutive), enhancers, transcription terminators, a start codon (i.e., ATG), splicing signals for introns, and stop codons.
  • “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
  • Amplification refers to any means by which a polynucleotide sequence is copied and thus expanded into a larger number of polynucleotide molecules, e.g., by reverse transcription, polymerase chain reaction, and ligase chain reaction.
  • Primer refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase.
  • a primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications.
  • a primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can,be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • Probe when used in reference to a polynucleotide, refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide.
  • a probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can'be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • a first sequence is an "antisense sequence" with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically hybridizes with a polynucleotide whose sequence is the second sequence.
  • Hybridizing specifically to or “specific hybridization” or “selectively hybridize to” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • a complex mixture e.g., total cellular DNA or RNA.
  • stringent conditions refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences.
  • Stringent hybridization and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York.
  • highly stringent hybridization and wash conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the Tm for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42° C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15 M NaCI at 72° C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2X SSC wash at 65° C for 15 minutes (see, Sambrook et al. for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1x SSC at 45° C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40° C for 15 minutes.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • the term “protein” typically refers to large polypeptides.
  • the term “peptide” typically refers to short polypeptides.
  • polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • Allelic variant refers to any of two or more polymorphic forms of a gene occupying the same genetic locus. Allelic variations arise naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. "Allelic variants” also refer to cDNAs derived from mRNA transcripts of genetic allelic variants, as well as the proteins encoded by them.
  • nucleotide or percent “identity,” in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • Antibody refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen).
  • the recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)'2 fragments.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies and humanized antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CH1, CH2 and CH3, but does not include the heavy chain variable region.
  • a ligand or a receptor e.g., an antibody "specifically binds to” or “is specifically immunoreactive with” a compound analyte when the ligand or receptor functions in a binding reaction which is determinative of the presence of the analyte in a sample of heterogeneous compounds.
  • assay e.g., immunoassay
  • a polynucleotide specifically binds under hybridization conditions to an analyte polynucledtide comprising a complementary sequence; an antibody specifically binds under immunoassay conditions to an antigen analyte bearing an epitope against which the antibody was raised; and an adsorbent specifically binds to an analyte under proper elution conditions.
  • Immunoassay refers to a method of detecting an analyte in a sample in which specificity for the analyte is conferred by the specific binding between an antibody and a ligand. This includes detecting an antibody analyte through specific binding between the antibody and a ligand. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • substantially pure or “isolated” means an object species is the predominant species present (i.e., on a molar basis, more abundant than any other individual macromolecular species in the composition), and a substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present.
  • a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition is the purified species of interest.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species.
  • Solvent species, small molecules ( ⁇ 500 Daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition.
  • Naturally-occurring refers to the fact that the object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • Detecting refers to determining the presence, absence, or amount of an analyte in a sample, and can include quantifying the amount of the analyte in a sample or per cell in a sample.
  • Detectable moiety refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
  • the detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantitate the amount of bound detectable moiety in a sample.
  • the detectable moiety can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin.
  • the detectable moiety may be directly or indirectly detectable. Indirect detection can involve the binding of a second directly or indirectly detectable moiety to the detectable moiety.
  • the detectable moiety can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize.
  • the binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule.
  • the binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules. (See, e.g., PD. Fahrlander and A. Klausner, Bio / Technology (1988) 6:1165.) Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.
  • a "subject" of diagnosis or treatment is a human or non-human mammal.
  • Non-human mammals subject to diagnosis or treatment include, for example, primates, ungulates, canines and felines.
  • Diagnostic means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives).
  • the "specificity” of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • Prognostic means predicting the probable development (e.g., severity) of a pathologic condition.
  • Test amount refers to an amount of an analyte in a subject sample, which is then compared to a normal amount of the analyte in a sample (e.g., from a healthy individual) such that the relative comparison of the values provides a reference value for diagnosing a designated disease.
  • the test amount may be a determination of the amount of the analyte, but it is not necessarily an amount.
  • the test amount may also be a relative value, such as a plus or a minus score, and also includes an amount indicating the presence or absence of the analyte in a sample.
  • Normal amount refers to an amount or a range of an analyte in a biological sample that indicates health or lack of pathology.
  • Diagnostic amount refers to an amount of an analyte in a subject sample that is consistent with a particular diagnosis for a designated disease.
  • Prognostic amount refers to an amount or range of an analyte in a subject sample that is consistent with a particular prognosis for a designated disease.
  • An “epitope” is portion of a molecule that specifically binds to an antibody or a T cell receptor.
  • a peptide epitope generally comprises a sequence of at least 6 amino acids from a polypeptide, although longer and shorter peptides can constitute epitopes.
  • Repro-EN-1.0 The autoantigen is called Repro-EN-1.0.
  • the presence of antibodies that specifically bind to an epitope of the Repro-EN-1.0 polypeptide is a highly sensitive and specific diagnostic marker for endometriosis.
  • Polynucleotides encoding full-length Repro-EN-1.0 are useful in recombinant production of Repro-EN-1.0.
  • the Repro-EN-1.0 polypeptide is useful as a positive control in diagnostic assays to detect antibodies that specifically bind to Repro-EN-1.0 from patient serum samples.
  • the polypeptide is also useful as an immunogen for eliciting production of antibodies against epitopes of the protein.
  • This 3164-base nucleotide sequence contains an open reading frame of 2580 nucleotides encoding Repro-EN-1.0 from nucleotide 176 to nucleotide 2755.
  • the deduced amino acid sequence of Repro-EN-1.0 has 860 amino acids.
  • Repro-EN-1.0 has a calculated molecular mass of 96.4 kD and a pI of 5.08.
  • the Repro-EN-1.0 gene encodes a 3.4 kb mRNA. This mRNA is expressed primarily in skeletal muscle, heart and testis, and to a lesser extent in other tissues. However, it is not detected in lung or peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • IB1 an alternately spliced variant that was isolated form a human heart cDNA library.
  • This variant is called IB1 and is useful in the same ways as the nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of Repro-EN-1.0.
  • the IB1 sequence was isolated from a heart cDNA library by screening with a nucleic acid probe obtained from the Repro-EN-1.0 sequence.
  • the IB1 sequence is different from the Repro-EN-1.0 sequence in that it contains an additional 231 bp exon inserted into the cDNA sequence at position 1555. Therefore the IB1 sequence has similar properties, but is slightly larger.
  • IB1 Alternately Spliced Variant from Heart Library:
  • amino acid sequence identified several amino acid motifs that will be apparent to those skilled in the art including a myb 1 DNA binding domain, a WD 40 site, an RGD cell-attachment sequence, an N-myristolylation site and several phosphorylation and glycosylation sites. Analysis also shows that the protein is largely hydrophilic. This implies that most of the amino acid sequence is exposed to the immune system and can be recognized as epitopes.
  • ESTs expressed sequence tags
  • This invention provides recombinant polynucleotides comprising a nucleotide sequence encoding Repro-EN-1.0 and IB1 proteins as described herein.
  • the polynucleotides are useful for expressing the mRNA or polypeptides they encode and in the preparation of probes or primers, among other things.
  • the recombinant polynucleotide molecule comprises a nucleotide sequence encoding polypeptide Repro-EN-1.0 of SEQ ID NO:2 or IB1 of SEQ ID NO:4.
  • the nucleotide sequence can be identical to a sequence from Repro-EN-1.0 cDNA or its complement or IB1 cDNA or its complement, or can include degenerate codons.
  • the sequence is identical to the coding sequence of Repro-EN-1.0 of SEQ ID NO:1 or IB1 of SEQ ID NO:3.
  • the polynucleotide encodes a fusion protein between Repro-EN-1.0 or IB1 polypeptide or Repro-EN-1.0 or IB1 analog amino acid sequences and a second amino acid sequence.
  • the second amino acid sequence can be, for example, a detectable label such as a fluorescent protein, enzyme marker of protein from a two-hybrid system.
  • the polynucleotides of the present invention are cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Q ⁇ replicase amplification system (QB).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustained sequence replication system
  • QB Q ⁇ replicase amplification system
  • a polynucleotide encoding the protein can be isolated by polymerase chain reaction of cDNA from a human endometrial carcinoma cell line using primers based on the DNA sequence of Repro-EN-1.0 of SEQ ID NO:1.
  • One pair of primers useful for amplifying Repro-EN-1.0 DNA, including allelic variants, is:
  • PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol . 51:263; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).
  • One useful format is real time PCR. See, e.g., Luch et al. (1997) J. Molec. Endocrinol . 18:77-85 and Arold et al., (1997) Proc. Nat'l Acad. Sci., USA 94:2438-43.
  • Polynucleotides also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of SEQ ID NO:1 under stringent hybridization conditions.
  • Mutant versions of the proteins can be made by site-specific mutagenesis of other polynucleotides encoding the proteins, or by random mutagenesis caused by increasing the error rate of PCR of the original polynucleotide with 0.1 mM MnCl2 and unbalanced nucleotide concentrations.
  • This invention also provides expression vectors, e.g., recombinant polynucleotide molecules comprising expression control sequences operatively linked to a nucleotide sequence encoding the target polypeptide.
  • Expression vectors can be adapted for function in prokaryotes or eukaryotes by inclusion of appropriate promoters, replication sequences, markers, etc. for transcription and translation of mRNA.
  • the construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning techniques also well known in the art. Sambrook et al., Molecular Cloning - A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) and Current Protocols in Molecular Biology, F.M.
  • Useful promoters for such purposes include a metallothionein promoter, a constitutive adenovirus major late promoter, a dexamethasone-inducible MMTV promoter, a SV40 promoter, a MRP polIII promoter, a constitutive MPSV promoter, a tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), and a constitutive CMV promoter.
  • Expression vectors useful in this invention depend on their intended use. Such expression vectors must, of course, contain expression and replication signals compatible with the host cell.
  • Expression vectors useful for expressing the protein of this invention include viral vectors such as alpha viruses, retroviruses, adenoviruses and adeno-associated viruses, plasmid vectors, cosmids, liposomes and the like. Viral and plasmid vectors are preferred for transfecting mammalian cells.
  • the expression vector pcDNA1 Invitrogen, San Diego, CA), in which the expression control sequence comprises the CMV promoter, provides good rates of transfection and expression.
  • Adeno-associated viral vectors are useful in the gene therapy methods of this invention.
  • the construct can also contain a tag to simplify isolation of the protein.
  • a polyhistidine tag of, e.g., six histidine residues, can be incorporated at the amino terminal end of the protein.
  • the polyhistidine tag allows convenient isolation of the protein in a single step by nickel-chelate chromatography.
  • endogenous genes are transcribed by operatively linking them to expression control sequences supplied endogenously that recombine with genomic DNA.
  • one provides the cell with a recombinant polynucleotide containing a targeting sequence, which permits homologous recombination into the genome upstream of the transcriptional start site of target gene; the expression control sequences; an exon of the target gene; and an unpaired splice-donor site which pairs with a splice acceptor in the target gene.
  • a targeting sequence which permits homologous recombination into the genome upstream of the transcriptional start site of target gene; the expression control sequences; an exon of the target gene; and an unpaired splice-donor site which pairs with a splice acceptor in the target gene.
  • the invention also provides recombinant cells comprising an expression vector for expression of the nucleotide sequences encoding a polypeptide of this invention.
  • Host cells can be selected for high levels of expression in order to purify the protein. Mammalian cells are preferred for this purpose, but prokaryotic cells, such as E. coli, also are useful.
  • the cell can be, e.g., a recombinant cell in culture or a cell in vivo .
  • This invention provides polynucleotide probes and primers that specifically hybridize to a sub-sequence of Repro-EN-1.0 cDNA or its complement or IB1 cDNA or its complement, under stringent hybridization conditions.
  • the probes and primers of this invention are polynucleotides of at least 7 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides or at least 25 nucleotides.
  • the sequence of the polynucleotide is a contiguous sequence from SEQ ID NO: 1 or its complement. Any suitable region of the Repro-EN-1.0 or IB1 gene may be chosen as a target for polynucleotide hybridization.
  • Nucleotide substitutions, deletions, and additions may be incorporated into the polynucleotides as long as the characteristic ability to specifically hybridize to the target sequence or its complement is retained. Nucleotide sequence variation may result from sequence polymorphisms of various alleles, minor sequencing errors, and the like.
  • the probes and primers of the invention are useful as probes in hybridization assays, such as Southern and northern blots, for identifying polynucleotides having a nucleotide sequence encoding a Repro-EN-1.0 or IB1 polypeptide, and as primers for amplification procedures.
  • the probes and primers of the invention are also useful in detecting the presence, absence or amount of Repro-EN-1.0 or IB1 in tissue biopsies and histological sections where the detection method is carried out in situ, typically after amplification of Repro-EN-1.0 or IB1 sequences using a primer set.
  • probes and primers of this invention also are useful for identifying allelic forms of Repro-EN-1.0 and animal cognate genes or IB1 and animal cognate genes.
  • Probes and primers can be used to screen human or animal genomic DNA or cDNA libraries under, e.g., stringent conditions. DNA molecules that specifically hybridize to the probe are then further examined to determine whether they are Repro-EN-1.0 allelic variants or animal cognates or IB1 allelic variants or animal cognates.
  • the probes also are useful in oligonucleotide arrays. Such arrays are used in hybridization assays to check the identity of bases in a target polynucleotide. In essence, when a target hybridizes perfectly to a probe on the array, the target contains the nucleotide sequence of the probe. When the target hybridizes less well, or does not hybridize at all, then the target and probe differ in sequence by one or more nucleotide. By proper selection of probes, one can check bases on a target molecule. See, e.g., Chee et al., WO 95/11995. The use the Repro-EN-1.0 or IB1 sequence in genomics is described further below.
  • the polynucleotide is directly or indirectly detectable through a detectable moiety.
  • a detectable moiety bound to either an oligonucleotide primer or a probe is subsequently used to detect hybridization of an oligonucleotide primer to the RNA component. Detection of labeled material bound to a Repro-EN-1.0 or IB1 polynucleotide in a sample provides a means of determining a diagnostic or prognostic value.
  • primers and probes can differ in sequence and length, the primary differentiating factor is one of function: primers serve as an initiation point for DNA synthesis of a target polynucleotide, as in RT and PCR reactions, while probes are typically used for hybridization to and detection of a target polynucleotide. Typical lengths of primers or probes can range from 7-50 nucleotides, preferably from 10-40 nucleotides, and most preferably from 15-35 nucleotides. A primer or probe can also be labeled with a detectable moiety for detection of hybridization of the primer or probe to the target polynucleotide.
  • polynucleotides used in the invention include both DNA and RNA molecules and naturally occurring modifications thereof, as well as synthetic, non-naturally occurring analogs of the same, and heteropolymers, of deoxyribonucleotides, ribonucleotides, and/or analogs of either.
  • the particular composition of a polynucleotide or polynucleotide analog will depend upon the purpose for which the material will be used and the environment in which the material will be placed. Modified or synthetic, non-naturally occurring nucleotides have been designed to serve a variety of purposes and to remain stable in a variety of environments, such as those in which nucleases are present.
  • Oligonucleotides preferably are synthesized, e.g., on an Applied BioSystems or other commercially available oligonucleotide synthesizer according to specifications provided by the manufacturer. Oligonucleotides may be prepared using any suitable method, such as the phosphotriester and phosphodiester methods, or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidates are used as starting materials and may be synthesized as described by Beaucage et al., Tetrahedron Letters 22: 1859 (1981), and U.S. Patent No. 4,458,066.
  • Polynucleotides e.g., probes, also can be recombinantly produced through the use of plasmids or other vectors.
  • this invention provides a probe that specifically hybridizes to the 5' untranslated region of Repro-EN-1.0 or IB1, the coding region of Repro-EN-1.0 or IB1, or a region of Repro-EN-1.0 or IB1 encoding an epitope of the Repro-EN-1.0 or IB1 polypeptide.
  • this invention provides a primer pair which amplifies a nucleotide sequence encoding a polypeptide epitope of Repro-EN-1.0 or IB1 recognized by an antibody from an individual diagnosed with endometriosis.
  • a primer pair that amplifies a particular nucleotide includes a 5' primer and a 3' primer.
  • the 3' primer hybridizes to the 3' end of the nucleotide sequence or downstream from it.
  • the 5' primer hybridizes to the 3' end of the complement of the nucleotide sequence or downstream from it.
  • the primers can amplify a polynucleotide that comprises the nucleotide sequence.
  • One nucleotide sequence encoding a polypeptide epitope of Repro-EN-1.0 has been identified within the about 2.2 kb from 3' end of the coding sequences of Repro-EN-1.0 (SEQ ID NO:1).
  • This invention also provides purified, recombinant Repro-EN-1.0 and IB1 polypeptide.
  • the invention provides recombinant Repro-EN-1.0 polypeptides which include the polypeptide whose amino acid sequence is presented in SEQ ID NO:2, as well as allelic variants of it.
  • Repro-EN-1.0 polypeptide refers to native Repro-EN-1.0, the polypeptide whose amino acid sequence is the amino acid sequence of SEQ ID NO:2, and to allelic variants of it.
  • Polynucleotide molecules that encode allelic variants of Repro-EN-1.0 are isolatable from endometrial cancer cell cDNA or genomic DNA and typically hybridize under stringent conditions to the nucleotide sequence encoding Repro-EN-1.0 (SEQ ID NO:1). They can be obtained by amplification using, e.g., PCR primers taken from the sequence of Repro-EN-1.0 described herein.
  • Repro-EN-1.0 polypeptides are useful as immunogens to elicit the production of anti-Repro-EN-1.0 antibodies, as affinity capture molecules to isolate such antibodies from a mixture, and as controls in diagnostic methods aimed at detecting Repro-EN-1.0 in a sample.
  • the cDNA encoding Repro-EN-1.0 of SEQ ID NO:1 was discovered by screening an expression library of cDNA from an endometrial carcinoma cell line with serum pooled from subjects diagnosed with endometriosis. Therefore, polypeptides comprising an epitope of Repro-EN-1.0 can be identified by screening with such serum.
  • the test serum is a serum pooled from several subjects positively diagnosed with endometriosis.
  • At least one epitope of Repro-EN-1.0 exists in a fragment of 567 amino acids from the carboxy-terminus of the molecule (amino acids 293-860 of SEQ ID NO:2).
  • At least one epitope of IB1 exists in a fragment of 644 amino acids from the carboxy terminus of the molecule (amino acids 293-937 of SEQ ID NO: 4) to detect the presence of antibodies against Repro-EN-1.0 or IB1 in patient serum samples. This test is useful in diagnosis because the presence of such antibodies is a diagnostic marker.
  • Repro-EN-1.0 or IB1 are most easily produced recombinantly, as described herein.
  • Recombinant Repro-EN-1.0 or IB1 can be purified by affinity purification
  • recombinant Repro-EN-1.0 or IB1 comprise a polyhistidine tag.
  • the protein is purified on a nickel-chelate affinity matrix.
  • Repro-EN-1.0 or IB1 is purified using an affinity matrix carrying anti-Repro-EN-1.0 or IB1 antibodies.
  • this invention provides a composition comprising an antibody that specifically binds Repro-EN-1.0 or IB1 polypeptides.
  • Antibodies preferably have affinity of at least 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , or 10 9 M -1 .
  • This invention contemplates both polyclonal and monoclonal antibody compositions.
  • Immunotoxins against Repro-EN-1.0- or IB1-expressing cells are also described herein.
  • Immunotoxins are antibodies and the like as described herein coupled to a compound, e.g., a toxin, that is toxic to a target cell.
  • Toxins can include, for example, radioactive isotopes, ricin, cisplatin, antisense molecules, Diphtheria toxin, Pseudomonas exotoxin A or Bacillus anthraces protective antigen.
  • Immunotoxins bind to cancer cells that express Repro-EN-1.0 or IB1 and kill them. They are useful in the therapeutic methods of this invention.
  • the antibodies of the invention have many uses.
  • such antibodies are useful for detecting Repro-EN-1.0 or IB1 polypeptides in immunoassays.
  • the antibodies also can be used to screen expression libraries for particular expression products such as mammalian Repro-EN-1.0 or IB1. These are useful for detecting or diagnosing various pathological conditions related to the presence of the respective antigens.
  • the antibodies in such a procedure are labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
  • Antibodies raised against Repro-EN-1.0 or IB1 can also be used to raise anti-idiotypic antibodies.
  • a number of immunogens are used to produce antibodies that specifically bind Repro-EN-1.0 or IB1 polypeptides.
  • Full-length Repro-EN-1.0 or IB1 is a suitable immunogen.
  • the immunogen of interest is a peptide of at least about 3 amino acids, more typically the peptide is 5 amino acids in length, preferably, the fragment is 10 amino acids in length and more preferably the fragment is 15 amino acids in length or greater.
  • Thepeptides can be coupled to a carrier protein (e.g., as a fusion protein), or are recombinantly expressed in an immunization vector.
  • Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length. Naturally occurring polypeptides are also used either in pure or impure form.
  • Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells and purified using standard techniques.
  • the polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies.
  • Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.
  • an immunogen preferably a purified polypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a polypeptide incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Patent No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture.
  • an immunogen preferably a purified polypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a polypeptide incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Patent No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the poly
  • Antibodies including binding fragments and single chain recombinant versions thereof, against predetermined fragments of Repro-EN-1.0 or IB1 proteins are raised by immunizing animals, e.g., with conjugates of the fragments with carrier proteins as described above.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified polypeptides, or screened for agonistic or antagonistic activity, e.g., activity mediated through Repro-EN-1.0 or IB1. In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in, e.g., Stites et al.
  • recombinant immunoglobulins may be produced. See, Cabilly, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033.
  • polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature.
  • an antibody used for detecting an analyte can be directly labeled with a detectable moiety, or may be indirectly labeled by, for example, binding to the antibody a secondary antibody that is, itself directly or indirectly labeled.
  • the antibodies of this invention are also used for affinity chromatography in isolating Repro-EN-1.0 or IB1 proteins.
  • Columns are prepared, e.g., with the antibodies linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified Repro-EN-1.0 or IB1 polypeptides are released
  • Fragments of antibodies against Repro-EN-1.0 or IB1 protein or protein analogs are also described herein. Typically, these fragments exhibit specific binding to the Repro-EN-1.0 protein receptor similar to that of a complete immunoglobulin.
  • Antibody fragments include separate heavy chains, light chains Fab, Fab' F(ab') 2 and Fv. Fragments are produced by recombinant DNA techniques, or by enzymic or chemical separation of intact immunoglobulins
  • Repro-EN-1.0 or IB1 polypeptides can be identified by any methods known in the art.
  • the methods involve detecting the polypeptide with a ligand that specifically recognizes the polypeptide (e.g., an immunoassay).
  • the antibodies of the invention are particularly useful for specific detection of Repro-EN-1.0 or IB1 polypeptides.
  • a variety of antibody-based detection methods are known in the art. These include, for example, radioimmunoassay, sandwich immunoassays (including ELISA), immunofluorescent assays, western blot, affinity chromatography (affinity ligand bound to a solid phase), and in situ detection with labeled antibodies.
  • Another method for detecting Repro-EN-1.0 or IB1 polypeptides involves identifying the polypeptide according to its mass through, for example, gel electrophoresis, mass spectrometry or HPLC.
  • Subject samples can be taken from any number of appropriate sources, such as saliva, peritoneal fluid, blood or a blood product (e.g., serum), urine, tissue biopsy (e.g., lymph node tissue), etc.
  • the methods for detection of Repro-EN-1.0 or IB1 polypeptides may employ one or more anti-Repro-EN-1.0 or IB1 antibody reagents (i.e., immunoassays).
  • immunoassays i.e., immunoassays
  • a number of well established immunological binding assay formats suitable for the practice of the invention are known (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g., METHODS IN CELL BIOLOGY VOLUME 37: ANTIBODIES IN CELL BIOLOGY, Asai, ed. Academic Press, Inc.
  • immunological binding assays utilize a "capture agent" to specifically bind to and, often, immobilize the analyte to a solid phase.
  • the capture agent is a moiety that specifically binds to a Repro-EN-1.0 or IB1 polypeptide or subsequence, such as an anti-Repro-EN-1.0 or anti-IB1 antibody.
  • the Repro-EN-1.0 or IB1 polypeptide being assayed is detected directly or indirectly using a detectable label.
  • the particular label or detectable group used in the assay is usually not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody or antibodies used in the assay.
  • the label may be covalently attached to the capture agent (e.g., an anti-Repro-EN-1.0 or or anti-IB1 antibody), or may be attached to a third moiety, such as another antibody, that specifically binds to the Repro-EN-1.0 polypeptide.
  • the methods employ competitive and noncompetitive immunoassays for detecting Repro-EN-1.0 or IB1 polypeptides.
  • Non-competitive immunoassays are assays in which the amount of captured analyte (in this case Repro-EN-1.0 or IB1) is directly measured.
  • One such assay is a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the Repro-EN-1.0 or IB1 protein. See, e.g., Maddox et al., 1983, J. Exp. Med. 158:1211 for background information.
  • the capture agent e.g., an anti-Repro-EN-1.0 or anti-IB1 antibody
  • the capture agent is bound directly to a solid substrate where it is immobilized.
  • These immobilized antibodies then capture any Repro-EN-1.0 or IB1 protein present in the test sample.
  • the Repro-EN-1.0 or IB1 polypeptide thus immobilized can then be labeled, i.e., by binding to a second anti-Repro-EN-1.0 or IB1 antibody bearing a label.
  • the second anti-Repro-EN-1.0 or IB1 antibody may lack a label, but be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second antibody alternatively can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • the amount of Repro-EN-1.0 or IB1 protein present in the sample is measured indirectly by measuring the amount of an added (exogenous) Repro-EN-1.0 or IB1 displaced (or competed away) from a capture agent (e.g., anti-Repro-EN-1.0 or anti-IB1 antibody) by the Repro-EN-1.0 or IB1 protein present in the sample.
  • a capture agent e.g., anti-Repro-EN-1.0 or anti-IB1 antibody
  • a hapten inhibition assay is another example of a competitive assay.
  • Repro-EN-1.0 or IB1 protein is immobilized on a solid substrate.
  • a known amount of anti-Repro-EN-1.0 or anti-IB1 antibody is added to the sample, and the sample is then contacted with the immobilized Repro-EN-1.0 or IB1 protein.
  • the amount of anti-Repro-EN-1.0 or anti-IB1 antibody bound to the immobilized Repro-EN-1.0 or IB1 protein is inversely proportional to the amount of Repro-EN-1.0 protein present in the sample.
  • the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. In this aspect, detection may be direct, where the antibody is labeled, or indirect where the label is bound to a molecule that specifically binds to the antibody as described above.
  • the methods detect and quantify the presence of Repro-EN-1.0 or IB1 polypeptide in the sample by using an immunoblot (Western blot) format.
  • Another immunoassay is the so-called "lateral flow chromatography.”
  • a sample moves across a substrate by, e.g., capillary action, and encounters a mobile labeled antibody that binds the analyte forming a conjugate.
  • the conjugate then moves across the substrate and encounters an immobilized second antibody that binds the analyte.
  • immobilized analyte is detected by detecting the labeled antibody.
  • the solid surface may be a membrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g.
  • the desired component may be covalently bound or noncovalently attached through nonspecific bonding.
  • organic and inorganic polymers both natural and synthetic may be employed as the material for the solid surface.
  • Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinyldene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like.
  • Other materials which may be employed include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cements or the like.
  • substances that form gels such as proteins ( e .
  • g ., gelatins lipopolysaccharides, silicates, agarose and polyacrylamides
  • Polymers which form several aqueous phases such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable.
  • the splid surface is porous, various pore sizes may be employed depending upon the nature of the system.
  • Mass spectrometers can measure mass by determining the time required for an ionized analyte to travel down a flight tube and to be detected by an ion detector.
  • MALDI matrix-assisted laser desorption/ionization mass spectrometry
  • SEAC Surfaces Enhanced for Affinity Capture
  • the diagnostic and prognostic assays described herein can be carried out in various combinations and can also be carried out in conjunction with other diagnostic or prognostic tests.
  • the presence of a Repro-EN-1.0 or IB1 polypeptide can be used to determine the stage of the disease.
  • Tests that may provide additional information include microscopic analysis of biopsy samples, detection of antigens (e.g., cell-surface markers) associated with endometriosis (e.g., using histocytochemistry, FACS, or the like).
  • Repro-EN-1.0 or IB1 we have detected circulating antibodies against Repro-EN-1.0 or IB1 in the blood of women diagnosed with endometriosis. This supports the idea that endometriosis has an autoimmune component. Further, Repro-EN-1.0 or IB1 and auto-antibodies against Repro-EN-1.0 or IB1 represent two targets in the diagnosis of endometriosis.
  • Repro-EN-1.0 or IB1 that is shed into the peritoneal fluid of women with endometriosis is useful in methods of diagnosing endometriosis. These methods include detecting Repro-EN-1.0 or IB1 in a biological sample of a subject. Suitable samples include, without limitation, saliva, blood or a blood product (e.g., serum), urine, menstrual fluid, vaginal secretion and, in particular, peritoneal fluid. Repro-EN-1.0 or IB1 can be detected by any of the methods described herein. Any detection of Repro-EN-1.0 or IB1 above a normal range is a positive sign in the diagnosis of endometriosis.
  • a blood product e.g., serum
  • Repro-EN-1.0 or IB1 can be detected by any of the methods described herein. Any detection of Repro-EN-1.0 or IB1 above a normal range is a positive sign in the diagnosis of endometriosis.
  • this invention provides methods for diagnosing endometriosis in a subject by detecting in a sample from the subject a diagnostic amount of an antibody that specifically binds to Repro-EN-1.0 or IB1 polypeptide.
  • Suitable patient samples include, without limitation, saliva, blood or a blood product (e.g., serum), peritoneal fluid, urine, menstrual fluid, vaginal secretion.
  • the antibodies can be detected by any of the methods for detecting proteins described herein. However, sandwich type assays are particularly useful.
  • all antibodies are captured onto a solid phase, for example using protein A, and antibodies specific for Repro-EN-1.0 or IB1 are detected using a directly or indirectly labeled Repro-EN-1.0 or IB1 or polypeptide fragment of it having an epitope of Repro-EN-1.0 or IB1.
  • Repro-EN-1.0 or IB1 or an antigenic fragment of it can be used as the capture molecule and captured antibodies can be detected.
  • the diagnostic method involves specifically detecting IgE or IgG 4 antibodies that specifically recognize Repro-EN-1.0 or IB1.
  • Anti-human IgE antibodies and anti-human IgG 4 antibodies can be easily bought or made.
  • Compounds that regulate the expression of Repro-EN-1.0 and IB1 are candidates as therapeutic agents. This invention provides methods for determining whether a compound regulates (e.g., activates or inhibits) expression of Repro-EN-1.0 or IB1.
  • Methods for determining whether a compound regulates Repro-EN-1.0 or IB1 expression involve administering to a cell or a test animal having an expressible Repro-EN-1.0 or IB1 gene with the compound, and determining whether expression Repro-EN-1.0 or IB1 is altered.
  • the methods involve administering the compound to a culture comprising the cell or to a test animal that has cells expressing Repro-EN-1.0 or IB1, measuring the amount of the Repro-EN-1.0 or IB1 polynucleotide or polypeptide in a sample from the culture or the animal, and determining whether the measured amount is different than the amount in a sample from the culture or from the animal under control conditions (e.g., to which no compound has been administered).
  • Statistically significant (p ⁇ 0.05) differences between the amount measured from the test sample and from the control sample are recorded and indicate that the compound alters the amount of Repro-EN-1.0 or IB1 produced by the cell.
  • the compound to be tested can be selected from a number of sources. For example, combinatorial libraries of molecules are available for screening experiments. Using such libraries, thousands of molecules can be screened for regulatory activity. In one preferred embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e . g ., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al . (1991) Nature , 354: 84-88).
  • Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT.Publication No WO 91/19735, 26 Dec.
  • nucleic acid libraries nucleic acid libraries
  • peptide nucleic acid libraries see, e . g ., U.S. Patent 5,539,083
  • antibody libraries see, e . g ., Vaughn et al . (1996) Nature Biotechnology , 14(3): 309-314), and PCT/US96/10287)
  • carbohydrate libraries see , e . g ., Liang et al . (1996) Science , 274: 1520-1522, and U.S. Patent 5,593,853
  • small organic molecule libraries see, e . g ., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoids U.S.
  • Patent 5,569,588, thiazolidinones and metathiazanones U.S. Patent 5,549,974, pyrrolidines
  • U.S. Patents 5,525,735 and 5,519,134, morpholino compounds U.S. Patent 5,506,337, benzodiazepines 5,288,514, and the like).
  • this invention provides methods useful in detecting polymorphic forms of the Repro-EN-1.0 or IB1 gene.
  • the methods involve comparing the identity of a nucleotide or amino acid at a selected position within the sequence of a test Repro-EN-1.0 or IB1 gene with the nucleotide or amino acid at the corresponding position from the sequence of native Repro-EN-1.0 (SEQ ID NO:1) or IB1 (SEQ ID NO:3).
  • the comparison can be carried out by any methods known in the art, including direct sequence comparison by nucleotide sequencing, sequence comparison or determination by hybridization or ident fication of RFLPs.
  • the method involves nucleotide or amino acid sequencing of the entire test polynucleotide orpolypeptide, or a subsequence from it, and comparing that sequence with the sequence of native Repro-EN-1.0 or IB1.
  • the method involves identifying restriction fragments produced upon restriction enzyme digestion of the test polynucleotide and comparing those fragments with fragments produced by restriction enzyme digestion of native Repro-EN-1.0 or IB1 gene. Restriction fragments from the native gene can be identified by analysis of the sequence to identify restriction sites.
  • Another embodiment involves the use of oligonucleotide arrays.
  • the method involves providing an oligonucleotide array comprising a set of oligonucleotide probes that define sequences selected from the native Repro-EN-1.0 or IB1 sequence, generating hybridization data by performing a hybridization reaction between the target polynucleotide molecules and the probes in the set and detecting hybridization between the target molecules and each of the probes in the set and processing the hybridization data to determine nucleotide positions at which the identity of the target molecule differs from that of native Repro-EN-1.0 or IB1.
  • the comparison can be done manually, but is more conveniently done by a programmable, digital computer.
  • Poly A+ RNA isolated from RL95-2 was used as a template for first strand cDNA synthesis.
  • the poly A+ RNA was analyzed by denaturing gel electrophoresis and ranged in size from 0.2 to 10 Kb (10).
  • An oligo dT primer containing an internal, protected Xhol site was annealed in the presence of a nucleotide mixture containing 5-methyl dCTP, and extended with MMTV reverse transcriptase.
  • Second strand synthesis of the RL95-2 cDNA/RNA hybrid was completed by the addition of RNase H, DNA polymerase 1, and dNTPs to the first strand synthesis reaction.
  • Pfu DNA polymerase was used to blunt-end the double stranded RL95-2 cDNA followed by the ligation of EcoRl adapters.
  • the cDNA was kinased and digested with Xhol and EcoRl before size fractionation on Sephacryl S-500 columns, The size fractionated cDNA was recovered and the quantified on ethidium bromide containing plates against a set of serially-diluted DNA standards.
  • the cDNA contained in the first two column fractionations was directionally ligated, in the sense orientation, to Xhol / EcoRl -digested uniZAP phage vector arms. Initially, approximately 25 ng (per fraction) of the cDNA was ligated and packaged into bacteriophage particles. Subsequently, the approximately 100 ng remaining cDNA in fractions 1 and 2 was packaged into bacteriophage particles using several reactions of the lambda phage packaging extract (Stratagene).
  • the primary human RL95-2 library was titered by infection of the XL1 Blue host strain.
  • the ratio of recombinant:nonrecombinant phage was determined by plating infected XL1 Blue in the presence of IPTG and XGal.
  • the number of blue (non-recombinant) or white (recombinant) plaques were quantified using a Manostat colony counter.
  • Ninety-eight and one-half (98.5) percent of the phage in the human RL95-2 cDNA library were recombinant and the primary phage library base consisted of 2.2 x 10 6 independent clones.
  • the human RL95-2 cDNA library was amplified once and re-titered as above. The titer of the amplified library was 3 x 10 9 pfu/ml.
  • the average size of the cDNA inserts was determined by PCR amplification. Twenty well-isolated phage plaques were cored and the cDNA inserts were amplified using T7/T3 specific oligonucleotides which hybridize to sites flanking the cDNA insertion site contained within the lambda phage vector. The PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. Ninety--five percent of the cored plaques contained inserts varying in size from 0.5 to 2.5 Kb with an average size of 1.5 Kb.
  • the nucleotide sequence of Repro-EN-1.0 was determined by using a modified protocol of the dideoxy chain termination method of Sanger et al. and USB Sequenase 2.0 (Barker, D.F. 1993, Biotechniques ).
  • the amino acid sequence was predicted using the Intelligenetic TRANSLATE program and sequence homologies were determined with BLAST data base search algorithms.
  • the deduced amino acid sequence (in the expected frame for a fusion protein) was novel.
  • the Repro-EN-1.0 expression distribution was determined by Northern blot analysis using poly A+ RNA collected from human spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes (MTN human blot 11; Clontech) and human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (MTN human blot 1- Clontech) and the manufacturer' suggested protocols (Clontech).
  • MTN human blot 11 peripheral blood leukocytes
  • MTN human blot 1- Clontech human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas
  • the immobilized poly A+ RNA samples were incubated with a random prime labeled probe that represented the Repro-EN-1.0 insert.
  • the probe was generated by using and EcoRl / Xhol released fragment of pRepro-EN-1.0 as template for a random prime labeled reaction as described in the manufacturer's manual (Megaprime kit; Amersham).
  • the Northern blot membranes were prehybridized with 6 mls of ExpressHyb solution (Clontech) followed by a 1 hour incubation at 68°C with approximately 0.5 ng of radiolabeled probe (approx. 2X105 cpm) in 5 mls of ExpressHyb solution.
  • RNA species a 3.4 Kb mRNA, and tissue distribution was determined by autoradiography. Expression was detected primarily in skeletal muscle, heart and testis; and to a lesser extent in other tissues, but was not detected in lung or peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Repro-EN-1.0 was up-regulated in breast and uterine carcinomas relative to their normal counterparts, was highly expressed in both normal fallopian tube and fallopian tube carcinoma, and was expressed at low levels in both normal ovary and ovarian carcinoma (Fig. 2).
  • Expression of Repro-EN-1.0 in RL95-2 (endometrium carcinoma) cells is lower than in LNCaP (human prostate adenocarcinoma), PC-12 (rat cell line) and BT12 (human breast carcinoma cell line) cells and undetectable in a mouse hybridoma cell line (3E10; negative control) (Fig.3).
  • LNCaP human prostate adenocarcinoma
  • PC-12 rat cell line
  • BT12 human breast carcinoma cell line
  • Antibodies to peptides of the clone and/or to recombinant protein were generated in rabbits. This antisera was used to develop a Repro-EN-1.0 ELISA.
  • the predicted ORF for Repro-EN-1 was subcloned into an expression vector, and the recombinant protein was expressed and purified by Ni--Chelate chromatography using standard methodologies.
  • the purified recombinant Repro-EN-1 protein was used as a target antigen in an ELISA (EndXTM ELISA) designed to detect antigen-specific autoantibodies in patient serum.
  • ELISA EndXTM ELISA

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Claims (29)

  1. Rekombinantes Polynukleotid, umfassend eine Nukleotidsequenz, die das Polypeptid Repro-EN-1,0 der SEQ ID Nr. 2 codiert, wobei das Polypeptid spezifisch an Antikörper von Patienten mit der Diagnose Endometriose bindet.
  2. Polynukleotid nach Anspruch 1, worin die Nukleotidsequenz gewählt ist aus der Repro-EN-1,0-Sequenz der SEQ ID Nr. 1.
  3. Polynukleotid nach Anspruch 1, worin die Nukleotidsequenz eine native Repro-EN-1,0-Nukleotidsequenz ist.
  4. Polynukleotid nach Anspruch 1, worin die Nukleotidsequenz identisch zu Nukleotiden 176 bis 2755 der SEQ ID Nr. 1 ist.
  5. Rekombinantes Polynukleotid, umfassend eine Nukleotidsequenz, die das Polypeptid IB1 der SEQ ID Nr. 4 codiert, wobei das Polypeptid spezifisch an Antikörper von Patienten mit der Diagnose Endometriose bindet.
  6. Polynukleotid nach Anspruch 5, wobei die Nukleotidsequenz gewählt ist aus der IB1-Sequenz von SEQ ID Nr. 3.
  7. Polynukleotid nach Anspruch 5, worin die Nukleotidsequenz eine native IB1-Nukleotidsequenz ist.
  8. Polynukleotid nach Anspruch 5, worin die Nukleotidsequenz identisch zu Nukleotiden 176 bis 2986 der SEQ ID Nr. 3 ist.
  9. Polynukleotid nach einem der Ansprüche 1 bis 8, außerdem umfassend eine Expressionskontrollsequenz, die operativ an die Nukleotidsequenz geknüpft ist.
  10. Polynukleotid-Primerpaar, welches eine Nukleotidsequenz amplifiziert, die das Polypeptid Repro-EN-1,0 der SEQ ID Nr. 2 oder das Polypeptid IB1 der SEQ ID Nr. 4 codiert, wobei das Polypeptid spezifisch an Antikörper von Patienten mit der Diagnose Endometriose bindet, welches Paar umfasst:
    (1) einen 3'-Primer von mindestens 7 Nukleotiden, der spezifisch an ein 3'-Ende der Nukleotidsequenz oder stromabwärts von der Sequenz hybridisiert, und
    (2) einen 5'-Primer von mindestens 7 Nukleotiden, der spezifisch an das 3'-Ende des Komplements der Nukleotidsequenz oder stromabwärts vom Komplement der Sequenz hybridisiert.
  11. Polynukleotid-Primerpaar nach Anspruch 10, worin der 3'-Primer eine Sequenz aufweist, die komplementär zu einer Nukleotidsequenz ist, gewählt aus Repro-EN-1,0-cDNA der SEQ ID Nr. 1, und der 5'-Primer eine Sequenz aufweist, die identisch zur Nukleotidsequenz ist, gewählt aus Repro-EN-1,0-cDNA der SEQ ID Nr. 1.
  12. Polynukleotid-Primerpaar nach Anspruch 10, worin der 3'-Primer eine Sequenz aufweist, die komplementär zu einer Nukleotidsequenz ist, gewählt aus IB1-cDNA der SEQ ID Nr. 3, und der 5'-Primer eine Sequenz aufweist, die identisch zur Nukleotidsequenz ist, gewählt aus IB1-cDNA der SEQ ID Nr. 3.
  13. Polynukleotid-Primerpaar nach Anspruch 10, wobei das Paar der Polynukleotide Peptid-Nukleinsäuren sind.
  14. Rekombinante Zelle, umfassend ein rekombinantes Polynukleotid nach Anspruch 9.
  15. Gereinigtes, rekombinantes Repro-EN-1,0-Polypeptid, dessen Aminosäuresequenz identisch zu der von SEQ ID Nr. 2 oder einer Allel-Variante der SEQ ID Nr. 2 ist.
  16. Gereinigtes, rekombinantes IB1-Polypeptid, dessen Aminosäuresequenz identisch zu der von SEQ ID Nr. 4 oder einer Allel-Variante der SEQ ID Nr. 4 ist.
  17. Gereinigtes Polypeptid, umfassend Repro-EN-1,0 der SEQ ID Nr. 2 oder IB1 der SEQ ID Nr. 4, wobei das Polypeptid spezifisch an Antikörper von Patienten mit der Diagnose Endometriose bindet.
  18. Zusammensetzung, die im wesentlichen aus einem Antikörper besteht, der spezifisch an Repro-EN-1,0-Polypeptid der SEQ ID Nr. 2 bindet, oder einem Antikörper, der spezifisch an IB1-Polypeptid der SEQ ID Nr. 4 bindet.
  19. Zusammensetzung nach Anspruch 18, worin der Antikörper monoklonal ist.
  20. Verfahren zur Diagnose von Endometriose bei einem Patienten, welches die folgenden Schritte umfasst:
    (a) Nachweisen einer Testmenge eines Antikörpers, der spezifisch an Repro-EN-1,0-Polypeptid bindet, dessen Aminosäuresequenz identisch zu der von SEQ ID Nr. 2 oder einer Allel-Variante von SEQ ID Nr. 2 ist, oder IB1-Polypeptid, dessen Aminosäuresequenz identisch zu der von SEQ ID Nr. 4 oder einer Allel-Variante von SEQ ID Nr. 4 ist, in einer Probe von dem Patienten; und
    (b) Vergleichen der Testmenge mit einem normalen Bereich des Antikörpers in einer Kontrollprobe von einem Patienten, der nicht an Endometriose leidet,
    wobei eine Testmenge oberhalb des normalen Bereichs eine positive Indikation in der Diagnose der Endometriose erbringt.
  21. Verfahren nach Anspruch 20, wobei der Schritt des Nachweisens das Einfangen des Antikörpers aus der Probe mit einem immobilisierten Repro-EN-1,0 oder IB1 und das Nachweisen des eingefangenen Antikörpers umfasst.
  22. Verfahren nach Anspruch 20, wobei der Schritt des Nachweisens das Einfangen des Antikörpers aus der Probe mit einem immobilisierten Anti-Immunglobulin-Antikörper und das Nachweisen des eingefangenen Antikörpers umfasst.
  23. Verfahren nach Anspruch 21, wobei der Schritt des Nachweisens des eingefangenen Antikörpers das Zusammenbringen des eingefangenen Antikörpers mit einem nachweisbaren Antikörper, der Immunglobuline spezifisch bindet, und das Nachweisen der Bindung zwischen dem eingefangenen Antikörper und dem nachweisbaren Antikörper umfasst.
  24. Verfahren nach Anspruch 22, wobei der Schritt des Nachweisens des eingefangenen Antikörpers das Zusammenbringen des eingefangenen Antikörpers mit Repro-EN-1,0 oder IB1 und das Nachweisen der Bindung zwischen dem eingefangenen Antikörper und dem Repro-EN-1,0 oder IB1 umfasst.
  25. Verfahren nach einem der Ansprüche 20 bis 24, wobei die Probe Blutserum umfasst.
  26. Verfahren nach einem der Ansprüche 20 bis 25, wobei der Antikörper ein IgG-Immunglobulin, ein IgE-Immunglobulin oder ein IgG4-Immunglobulin ist.
  27. Verfahren zur Anwendung beim Verfolgen des Verlaufs einer Endometriose in einem Patienten, welches die folgenden Schritte umfasst:
    (a) Nachweisen erster und zweiter Mengen eines Antikörpers, der spezifisch Repro-EN-1,0-Polypeptid bindet, dessen Aminosäuresequenz identisch zu der von SEQ ID Nr. 2 oder einer Allel-Variante von SEQ ID Nr. 2 ist, oder ein IB1-Polypeptid, dessen Aminosäuresequenz identisch zu der von SEQ ID Nr. 4 oder einer Allel-Variante von SEQ ID Nr. 4 ist, in Proben von dem Patienten von einem ersten bzw. einem zweiten Zeitpunkt; und
    (b) Vergleichen der ersten und zweiten Mengen;
    wobei ein Anstieg zwischen den ersten und zweiten Mengen ein Fortschreiten der Endometriose indiziert und eine Abnahme zwischen den ersten und zweiten Mengen eine vorübergehende Besserung der Endometriose indiziert.
  28. Screening-Methode zum Bestimmen, ob eine Verbindung die Expression von Repro-EN-1,0 der SEQ ID Nr. 2 oder IB1 der SEQ ID Nr. 4 oder einer Allel-Variante davon in einer Zelle vermehrt oder vermindert, umfassend das Zusammenbringen der Zelle mit der Verbindung und das Bestimmen, ob die Produktion von Repro-EN-1,0- oder IN1-mRNA oder -Polypeptid vermehrt oder vermindert ist.
  29. Verfahren zum Detektieren polymorpher Formen von Repro-EN-1,0 der SEQ ID Nr. 1 oder 2 oder von IB1 der SEQ ID Nr. 3 oder 4, welches die folgenden Schritte umfasst:
    (a) Bestimmen der Identität eines Nukleotids oder Aminosäure an einer ausgewählten Position innerhalb der Sequenz eines Test-Repro-EN-1,0-Gens oder - Polypeptids oder eines Test-IB1-Gens oder -Polypeptids; und
    (b) Bestimmen der Identität des Nukleotids oder Aminosäure an einer entsprechenden Position des nativen Repro-EN-1,0-Gens oder -Polypeptids der SEQ ID Nr. 1 oder 2 oder des nativen IB1-Gens oder -Polypeptids der SEQ ID Nr. 3 oder 4; und
    (c) Vergleichen der Identität des Test-Gens oder -Polypeptids mit der Identität des nativen Gens oder Polypeptids, wobei ein Unterschied in der Identität anzeigt, dass das Test-Gens oder-polypeptids eine polymorphe Form von Repro-EN-1,0 oder IB1 ist.
EP00310408A 1999-11-23 2000-11-23 Endometriose-assoziierte-Autoantigene kodierendes Polynukleotid Expired - Lifetime EP1106690B1 (de)

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US8674932B2 (en) * 1996-07-05 2014-03-18 Anascape, Ltd. Image controller
US20030099976A1 (en) * 2001-01-17 2003-05-29 Tai-Jay Chang Androgen receptor complex-associated protein
US20030082721A1 (en) * 2001-01-17 2003-05-01 Tai-Jay Chang Transgenic animals expressing androgen receptor complex-associated protein
US6974683B2 (en) 2001-01-17 2005-12-13 Veterans General Hospital Nucleic acid encoding androgen receptor complex-associated protein
US7268117B2 (en) * 2003-07-11 2007-09-11 Praecis Pharmaceuticals Incorporated Compositions, kits, and methods for identification, assessment, prevention, and therapy of endometriosis
US20050181513A1 (en) * 2004-02-18 2005-08-18 Viorica Lopez-Avila Methods and compositions for assessing a sample by MAILDI mass spectrometry
US20060008876A1 (en) 2004-07-07 2006-01-12 Shami A S E ME-5, ME-2, and EPP2: human protein antigens reactive with autoantibodies present in the serum of women suffering from endometriosis
US8932993B1 (en) 2007-06-11 2015-01-13 Juneau Biosciences, LLC. Method of testing for endometriosis and treatment therefor
US11287425B2 (en) * 2009-04-22 2022-03-29 Juneau Biosciences, Llc Genetic markers associated with endometriosis and use thereof
DE102012002929A1 (de) 2012-02-14 2013-08-14 Jürgen Lewald Minimalinvasives Verfahren für die Diagnose und die Therapieverlaufskontrolle der Endometriose
US9434991B2 (en) 2013-03-07 2016-09-06 Juneau Biosciences, LLC. Method of testing for endometriosis and treatment therefor

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